• YAKHAK HOEJI
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• v.59 no.5
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• pp.207-214
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• 2015

P2Y receptors, a type of P2 receptor family, are G-protein coupled receptors and 8 subtypes have been characterized ($P2Y_1$, $P2Y_2$, $P2Y_4$, $P2Y_6$, $P2Y_{11-14}$). Recently, several studies have shed light on the role of P2Y receptors in bone biology. Among them, little is known on the role of $P2Y_6$ receptor on osteoclast differentiation. Thus, we investigated the role of $P2Y_6$ receptor on osteoclastogenesis using $P2Y_6$ receptor selective antagonist, MRS 2578. When osteoblasts and bone marrow cells were co-cultured in the presence of $VitD_3$ and $PGE_2$, $P2Y_6$ antagonist increased the formation of TRAP positive osteoclasts. To elucidate the target cells of $P2Y_6$ antagonist, we first checked the effect of MRS 2578 on osteoblasts. Treatment of MRS 2578 did not affect OPG : RANKL mRNA ratio in osteoblasts. Next, we checked the effects of $P2Y_6$ antagonist on osteoclast precursors using mouse bone marrow macrophages (BMMs). Addition of MRS 2578 increased the number of osteoclasts in RANKL-treated BMMs. Although $P2Y_6$ antagonist had no effect on RANKL-induced NFATc1, c-Fos and MafB expression levels, it significantly stimulated RANKL-induced Blimp1 mRNA expression in BMMs. Taken together, these data indicate that $P2Y_6$ antagonist increases osteoclast formation by upregulation of Blimp1 expression.

• Molecules and Cells
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• v.42 no.2
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• pp.123-134
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• 2019

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as $LPA_{1-6}$. For one of its receptors, $LPA_1$ (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

• BMB Reports
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• v.44 no.3
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• pp.147-157
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• 2011

The meiotic process from the primordial stage to zygote in female germ cells is mainly adjusted by post-transcriptional regulation of pre-existing maternal mRNA and post-translational modification of proteins. Several key proteins such as the cell cycle regulator, Cdk1/cyclin B, are post-translationally modified for precise control of meiotic progression. The second messenger (cAMP), kinases (PKA, Akt, MAPK, Aurora A, CaMK II, etc), phosphatases (Cdc25, Cdc14), and other proteins (G-protein coupled receptor, phosphodiesterase) are directly or indirectly involved in this process. Many proteins, such as CPEB, maskin, eIF4E, eIF4G, 4E-BP, and 4E-T, post-transcriptionally regulate mRNA via binding to the cap structure at the 5' end of mRNA or its 3' untranslated region (UTR) to generate a closed-loop structure. The 3' UTR of the transcript is also implicated in post-transcriptional regulation through an association with proteins such as CPEB, CPSF, GLD-2, PARN, and Dazl to modulate poly(A) tail length. RNA interfering is a new regulatory mechanism of the amount of mRNA in the mouse oocyte. This review summarizes information about post-transcriptional and post-translational regulation during mouse oocyte meiotic maturation.

• BMB Reports
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• v.29 no.6
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• pp.525-529
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• 1996

Ligand binding properties of muscarinic acetylcholine receptors (mAChRs) in the nematode Caenorhabditis elegans (C. elegans) were characterized by using filtration binding assays. Scatchard analysis using $[^{3}H]N-methylscopolamine$ ($[^{3}H]NMS$) showed that the dissociation constant ($K_d$) and the maximum binding value ($B_{max}$) were $3.3{\pm}0.8{\times}10^{10}$ M and $9.0{\pm}1.1$ fmol/mg protein, respectively. Binding competition experiments indicated that the affinities of C. elegans mAChRs to atropine, scopolamine, and oxotremorine were similar to those of mammalian mAChRs. Pirenzepine binding experiments revealed that the binding pattern of mAChRs in C. elegans closely resembled that of mAChRs in rat brain, suggesting that the receptors consist primarily of Ml subtype. The affinity of mAChRs for oxotrernorine was significantly affected by guanylylimidodiphosphate (Gpp(NH)p), a non hydrolyzable GTP analog, suggesting that mAChRs in C. elegans might be coupled to G proteins. The data presented here indicate the possibility that C. elegans provides a living animal model to study the action mode of the muscarinic cholinergic system.

• Development and Reproduction
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• v.9 no.1
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• pp.1-5
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• 2005

The hypothalamo-pituitary-gonadal hormone axis is centrally controlled by a complex regulatory network of excitatory and inhibitory signals, that is dormant during infantile and juvenile periods and activated at puberty. The kisspeptins are the peptide products of the KiSS-1 gene and the endogenous agonists for the G protein-coupled receptor 54(GPR54). Although KiSS-1 was initially discovered as a metastasis suppressor gene, a recent evidence suggests the KiSS-1/GPR54 system is a key regulator of the reproductive system. Yet the actual role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion remains largely unexplored, the system could be the first missing link in the reproductive hormonal axis. Central or peripheral administration of kisspeptin stimulates the hypothalamic-pituitary-gonadal axis, increasing circulating gonadotropin levels in rodents, sheep, monkey and human models. These effects appear likely to be mediated via the hypothalamic GnRH neuron system, although kisspeptins may have direct effects on the anterior pituitary gland. The loss of function mutations of the GPR54(GPR54-/-) have been associated with lack of puberty onset and idiopathic hypogonadotropic hypogonadism(IHH). So kisspeptin infusion may provide a novel mechanism for HPG axis manipulation in disorders of the reproductive system.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.155-162
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• 2020

Equine follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC₅₀ values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.147-152
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• 2009

Lysophosphatidic acid (LPA), a simple phospholipid-derived mediator implicated in diverse biological actions, acts through the specific G-protein coupled receptors, LPA receptor (LPAR) $1{\sim}5$. Our previous study showed that LPAR3 is expressed in the uterine endometrium in a cell type- and stage-specific manner and LPA via LPAR3 increases PTGS2 expression in the uterine endometrium during the period of implantation. Although LPAR3 is considered to be predominant LPA receptor in the uterine endometrium, other LPA receptors might playa role to mediate LPA functions in the uterine endometrium during pregnancy. Among LPARs, we investigated expression of LPAR1 during the estrous cycle and pregnancy in this study. Uterine endometrial tissue samples were collected from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Northern blot analysis determined that LPAR1 mRNA was constitutively expressed in the uterine endometrial tissues during the estrous cycle and pregnancy of all stages. Analysis by immunoblotting revealed that LPAR1 proteins were present in the porcine uterine endometrium during the estrous cycle and pregnancy. Immunohistochemical experiments demonstrated that LP AR1 protein was localized to endometrial epithelium and stromal cell, specifically to nuclei of these cell types. Results in this study show that LPAR1 is constitutively expressed in the uterine endometrium during the estrous cycle and pregnancy. These results suggest that LPA via LPAR1 may playa role in the uterine endometrial function throughout pregnancy in pigs.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.348-356
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• 2022

Background: Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin exerts its neuronal and non-neuronal in vitro and in vivo effects through LPA receptor subtypes. However, it is unknown whether gintonin can bind to the plasma membrane of cells and can transactivate the epidermal growth factor (EGF) receptor. In the present study, we examined whether gintonin-biotin conjugates directly bound to LPA receptors and transactivated the EGF receptor. Methods: We designed gintonin-biotin conjugates through gintonin biotinylation and examined whether gintonin-biotin conjugate binding sites co-localized with the LPA receptor subtype binding sites. We further examined whether gintonin-biotin transactivated the EGF receptor via LPA receptor regulation via phosphor-EGF and cell migration assays. Results: Gintonin-biotin conjugates elicit [Ca²⁺]_i transient similar to that observed with unbiotinylated gintonin in cultured PC3 cells, suggesting that biotinylation does not affect physiological activity of gintonin. We proved that gintonin-biotin conjugate binding sites co-localized with the LPA1/6 receptor binding sites. Gintonin-biotin binding to the LPA1 receptor transactivates the epidermal growth factor (EGF) receptor through phosphorylation, while the LPA1/3 receptor antagonist, Ki16425, blocked phosphorylation of the EGF receptor. Additionally, an EGF receptor inhibitor AG1478 blocked gintonin-biotin conjugate-mediated cell migration. Conclusions: We observed the binding between ginseng-derived gintonin and the plasma membrane target proteins corresponding to the LPA1/6 receptor subtypes. Moreover, gintonin transactivated EGF receptors via LPA receptor regulation. Our results suggest that gintonin directly binds to the LPA receptor subtypes and transactivates the EGF receptor. It may explain the molecular basis of ginseng physiology/pharmacology in biological systems.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.113-123
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• 2008

Microglia, which is the primary immune effector cells in the central nervous system, constitutes the first line of defense against infection and injury in the brain. The goal of this study was to determine the protective (anti-inflammation) mechanisms of forsythia suspense (FS) on LPS-induced activation of BV-2 microglial cells. The effects of FS on gene expression profiles in activated BV-2 microglial cells were evaluated using microarray analysis. BV-2 microglial cells were cultured in a 100mm dish $(1{\times}10^7/dish)$ for 24hr and then pretreated with $1{\mu}g/mL$ FS or left untreated for 30 min. Next, $1{\mu}g/mL$ LPS was added to the samples and the cells were reincubated at $37^{\circ}C$ for 30 min, 1hr, and 3hr. The gene expression profiles of the BV-2 microglial cells varied depending on the FS. The oligonucleotide microarray analysis revealed that MAPK pathway-related genes such as Mitogen activated protein kinase 1 (Mapk1), RAS protein activator like 2 (Rasal2), and G-protein coupled receptor 12 (Gpr12) and nitric oxide biosynthesis-related genes such as nitric oxide synthase 1 (neuronal) adaptor protein (Nos1ap), and dimethylarginine dimethylaminohydrolase 1 (Ddah1) were down regulated in FS-treated BV-2 microglial cells. FS can affect the MAPK pathway and nitric oxide biosynthesis in BV-2 microglial cells.

• Molecules and Cells
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• v.38 no.9
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• pp.796-805
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• 2015

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer's disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced $[Ca^{2+}]_i $ transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated $[Ca^{2+}]_i $ transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 1 2 weeks) also significantly attenuated amyloid-${\beta}$ protein ($A{\beta}$)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to $A{\beta}$ and could be utilized for AD prevention or therapy.